Polyclonal IgG was purified from human plasma samples collected on day 71, two weeks after the second immunization of either Ad26.COV2.S or placebo. This purification was conducted using NAb Protein G spin columns (Thermo Scientific). Protein G columns were preconditioned with Protein G IgG Binding Buffer (Thermo Scientific) and plasma samples were added to columns and incubated for 1 h. Columns were then washed, followed by elution with IgG Elution Buffer (Thermo Scientific). Eluted, purified IgG was buffer exchanged with 1 x DPBS and characterized by SDS-PAGE and spectrophotometry was used to quantify yield and normalize stocks to 10 mg-ml−1.
Protein g igg binding buffer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
Protein G IgG Binding Buffer is a solution used to facilitate the binding of IgG antibodies to Protein G, a bacterial protein that has a high affinity for the Fc region of IgG. This buffer optimizes the conditions for this interaction, enabling the efficient capture and purification of IgG from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Igg elution buffer, by Thermo Fisher Scientific (4 mentions)
Nab protein g spin columns, by Thermo Fisher Scientific (3 mentions)
Glycine, by Thermo Fisher Scientific (1 mentions)
Protein g coated sepharose beads, by GE Healthcare (1 mentions)
Whatman, by Cytiva (1 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (1 mentions)
6 protocols using protein g igg binding buffer
1
Purification of Polyclonal IgG from Human Plasma
2
Purification of Patient IgG
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Two millilitre protein G-coated sepharose beads (GE Healthcare Europe GmbH, Freiburg, Germany) were washed three times with PBS at 4 °, 300g for 5 min. Patient sera were three times diluted in protein G IgG binding buffer (BB; Thermo Fisher Scientific, Darmstadt, Germany), and then filtered using a 450 nm membrane (Whatman, Dessen, Germany). The sera were then transferred to a protein G column and incubated at room temperature (RT) for 1 h before extensive rinsing with 30 ml BB. Total IgG was eluted from the column with 0.1 M glycine, pH 2.7 and neutralized by Tris buffer pH 8.0 (Thermo Fisher Scientific, Darmstadt, Germany).
3
Rhesus Macaque IgG Purification
4
Purification of IgG from Serum
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IgG was purified from serum using a protein G-agarose column (2 ml of protein G-agarose beads). The columns were washed with protein G IgG binding buffer (catalog number 21019; Thermo Scientific), and bound IgG was subsequently eluted with 0.1 M glycine (pH 2 to 3) and immediately neutralized with 1 M Tris (pH 8). The different IgG eluate fractions were then pooled and buffer exchanged with 1× phosphate-buffered saline (PBS) using an Amicon Ultra 10K device spin column.
5
Purification of Mouse IgG Antibodies
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Mouse peripheral blood samples were prepared by centrifugation at 1,000 g for 15 min and diluted 1:15 with Protein G IgG binding buffer (Thermo Fisher Scientific Inc.). Diluted samples were loaded onto a Capturem Protein G Maxiprep Column (TaKaRa, Tokyo, Japan) and centrifuged at 2,000 g for 2 min. After washing the column with Protein G IgG binding buffer and PBS, the antibody was recovered using IgG Elution buffer (Thermo Fisher Scientific Inc.) and neutralized with pH neutralization buffer (1 M Tris, pH 9). Fractions containing the antibody were desalted using Slide‐A‐Lyzer MINI Dialysis device with a 10 kDa molecular weight cut‐off (Thermo Fisher Scientific Inc.). The purity of IgG preparations was analyzed by SDS‐PAGE.
6
Rhesus Macaque IgG Purification
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Polyclonal IgG was purified from rhesus macaque plasma using the NAb™ Protein G Spin Columns (Thermo Scientific). Plasm was incubated for 1 h with Protein G columns pre-conditioned with Protein G IgG Binding Buffer (Thermo Scientific) to capture maximum IgG. The columns were then washed six times, and IgG was eluted with IgG Elution Buffer (Thermo Scientific). The eluted IgG was buffer exchanged with 1x DPBS and analyzed by SDS-PAGE and spectrophotometry.
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